S. aureus (SA) causes a wide spectrum of clinical syndromes, and is the leading cause of endovascular infections world-wide. SA has a particular propensity to develop multi-drug resistance, and serious infections with such strains result in enhanced attributable mortalities. Since FDA approval in 2003, daptomycin (DAP) has been utilized in many clinical settings, especially for recalcitrant methicillin-resistant SA (MRSA) infections. There have been numerous recent reports of clinical SA strains that have evolved in vitro DAP-resistance in the context of failing DAP treatment regimens, especially in endovascular infections. One consistent feature of DAP-R strains is the acquisition of one or more gain-in-function mutations in a relatively restricted cadre of genes, especially mprF (multiple peptide resistance factor gene). This gene is responsible for the synthesis and translocation (flipping) of the SA-unique, positively-charged phospholipid (PL), lysyl-phosphotidylglycerol (L-PG) within its cell membrane (CM). Thus, mprF contributes substantially to the relative positive surface charge of SA. Moreover, in light of the absolute requirement for calcium association for DAP's bacterial lethality, genes such as mprF that impact surface charge are highly likely to be important in DAP-R, potentially via charge repulsion. A seminal feature of both clinical and in vitro-derived DAP-R SA strains is the frequent cross-resistance between DAP and cationic host defense peptides (HDPs) (13,15,17-19). Thus, our central hypothesis is that the development of DAP- R in MRSA is frequently associated with the co-evolution of HDP resistance, and this event impacts endovascular pathogenesis and treatment outcomes in vivo. We will address a number of important questions: i) how often do mprF gain-in-function mutations accompany DAP-HDP cross-resistance phenotypes?; ii) are such mprF mutations biased towards the synthase or flippase domains of this gene, and are they causal in cross-resistance?; iii) are there HDP- specific structural features that are shared amongst those peptides which exhibit the DAP-HDP cross-resistance phenotype?; iv) does the temporal exposure schedule of S. aureus strains to DAP and/or HDPs influence the development of cross-resistance?; and vi) what are the in vivo consequences of mprF mutations and DAP-HDP cross resistances upon innate virulence and responses to DAP therapy? We anticipate that these studies will contribute to a deeper understanding of the interactive role of our innate host defense system with exogenously administered antimicrobials in stimulating the adaptive survival response in SA. This should enable 'smart design' of future novel anti-SA agents that circumvent this adaptive response.